Development and Preclinical Testing of a High-Affinity Single-Chain Antibody against (+)-Methamphetamine

Abstract

Chronic or excessive (ϩ)-methamphetamine (METH) use often leads to addiction and toxicity to critical organs like the brain. With medical treatment as a goal, a novel single-chain variable fragment (scFv) against METH was engineered from anti-METH monoclonal antibody mAb6H4 (IgG, light chain, K d ϭ 11 nM) and found to have similar ligand affinity (K d ϭ 10 nM) and specificity as mAb6H4. The anti-METH scFv (scFv6H4) was cloned, expressed in yeast, purified, and formulated as a naturally occurring mixture of monomer (ϳ75%) and dimer (ϳ25%). To test the in vivo efficacy of the scFv6H4, male Sprague-Dawley rats (n ϭ 5) were implanted with 3-day s.c. osmotic pumps delivering 3.2 mg/kg/day METH. After reaching steady-state METH concentrations, an i.v. dose of scFv6H4 (36.5 mg/kg, equimolar to the METH body burden) was administered along with a [ 3 H]scFv6H4 tracer. Serum pharmacokinetic analysis of METH and [3 H]scFv6H4 showed that the scFv6H4 caused an immediate 65-fold increase in the METH concentrations and a 12-fold increase in the serum METH area under the concentration-time curve from 0 to 480 min after scFv6H4 administration. The scFv6H4 monomer was quickly cleared or converted to multivalent forms with an apparent t 1/2z of 5.8 min. In contrast, the larger scFv6H4 multivalent forms (dimers, trimers, etc.) showed a much longer t 1/2z (228 min), and the significantly increased METH serum molar concentrations correlated directly with scFv6H4 serum molar concentrations. Considered together, these data suggested that the scFv6H4 multimers (and not the monomer) were responsible for the prolonged redistribution of METH into the serum.There are currently nearly 20 monoclonal antibody (mAb) medications approved by the United States Food and Drug Administration and over 20 more in early clinical or preclinical trials (Holliger and Hudson, 2005). These medications include full-length IgG mAbs, along with five mAb fragments as Fab, F(abЈ) 2 (antigen binding fragments of IgG), or singlechain variable fragment (scFv) proteins.IgG mAbs are typically chimeric, humanized, or fully human proteins and are administered to patients requiring a long-acting antagonist with minimal extravascular penetration (Bazin-Redureau et al., 1997). IgG mAb is best for this purpose because it has a terminal elimination half-life (t 1/2z ) ranging from approximately 3 to 26 days. The longest t 1/2z values are usually achieved when the antibody does not bind to tissue sites and is not prematurely cleared due to antigenicity. When a short duration of action and greater extravascular penetration are needed, a significantly smaller fragment like Fab (t 1/2z ranging from 0.5-21 h; Trang, 1992) or scFv (t 1/2z ranging from minutes to hours; Goel et al., 2000) is used. In particular, rat pharmacokinetic studies of an antianthrax toxin scFv report a t 1/2␣ of ϳ5 min (Maynard et al., 2002). In addition, PCKN studies of a scFv in mice report t 1/2␣ values of 2.7 and 1 min Willuda et al., 2001). It is possible that a short-acting scFv could be used to

Crystal structures of a therapeutic single chain antibody in complex with two drugs of abuse—Methamphetamine and 3,4‐methylenedioxymethamphetamine

“…10 In an attempt to understand the mechanism of this stereo-specificity, we generated an in silico model for (À)-METH binding to scFv6H4 such that the interactions of the aromatic ring and hydrogen bonding interactions of the cationic nitrogen are retained. In this scenario, the methyl group attached to the chiral center of (À)-METH is too close to the Cb atom of TrpL91, suggesting that steric hindrances weaken the binding interactions for (À)-METH (Fig.…”

“…10 The scFv6H4 is comprised of a variable light chain domain (V L ) and a variable heavy chain (V H ) domain, both possessing immunoglobulin fold [ Fig. 4(A)].…”

“…10 The scFv6H4 was constructed by joining the light and heavy chain variable domains of the parent mAb6H4 (GenBank accession nos. DQ381543 and DQ381542, respectively) with a 15 amino acid linker of (Gly 4 Ser) 3 [ Fig.…”

“…ScFv6H4 was produced from the parent IgG without any loss of affinity or specificity. 10 In this report, we present the crystal structures of this high affinity scFv in complex with METH and MDMA.…”

“…Moreover, in therapeutic applications, when a short duration of action and greater extravascular penetration are needed, scFvs are better suited as they have a much shorter half life ranging from minutes to few hours. 9,10 Therefore, scFvs can be of great value in overdose cases when rapid removal of the drug from the body is necessary. 11 We crystallized a scFv derived from one of our highest affinity antibodies, mAb6H4 (K D ¼ 10 nM).…”